Process to manufacture implants containing bioactive peptides

ABSTRACT

A process for manufacturing a pharmaceutical composition for the delivery of an effective amount of a bioactive peptide or peptide analog over a period of 1 to 12 months. This process includes the steps of grinding a copolymer of lactic acid and glycolic acid having a ratio of glycolide to lactide units of from about 0 to 5:1 to a particle size of between about 50 and 150 μm; sterilizing the ground copolymer with a dose of between about 1 and 2.5 Mrads of ionizing γ-radiation; wetting the ground and sterilized copolymer with a sterile aqueous slurry of a bioactive peptide or peptide analog; aseptically blending the copolymer and the slurry to obtain a homogeneous mixture of the copolymer and between about 10 and 50% of the bioactive peptide or peptide analog; drying the mixture at reduced pressure and at temperature not exceeding 25° C.; aseptically extruding the dried mixture at a temperature between about 70 and 110° C.; and aseptically cutting cylindrical rods of about 1 to 2 mm diameter and between about 10 and 25 mm in length from the extruded mixture to form the pharmaceutical implants.

This application claims benefit of provisional application 60/025,449filed Sep. 4, 1996.

TECHNICAL FIELD

The invention relates to a novel process for preparing implants ofbioactive peptides or peptide analogs where such implants have a moreuniform distribution of peptide or peptide analog therein.

BACKGROUND ART

A wide variety of bioactive peptides and peptide analogs have been usedas active agents for the treatment of various conditions. These activeagents are generally administered in connection with a polymericdelivery system to control the release of the agent. For example,peptide analogs of the natural hypothalamic hormone LHRH (LuteinizingHormone Releasing Hormone, a decapeptide) are of therapeutic value whenadministered for a prolonged period of time with the appropriatedelivery system. Commercially successful delivery systems includemicrospheres, microcapsules, microgranules and other implant formswhich, when injected subcutaneously or intramuscularly, release the LHRHanalog from a biocompatible and biodegradable matrix. The matrix isfrequently a copolymer of lactic and glycolic acid ("PLGA", polylacticglycolic acid) as described, for example, in U.S. Pat. Nos. 3,773,919,3,887,499, 4,675,189, 4,767,628 and many others.

It has been assumed that a continuous or monophasic release of suchbioactive agents is a highly desirable feature of such formulations(see, e.g., U.S. Pat. No. 5,366,734). In fact, it has now been realizedthat what is really needed is to have the "therapeutic" effect of thepeptide or peptide analog be maintained or sustained over a relativelylong span of time (e.g., three to six months or longer). Thus,improvements in this area are desired and necessary.

SUMMARY OF THE INVENTION

The present invention relates to a process for manufacturingpharmaceutical implants for the delivery of an effective amount of abioactive peptide or peptide analog over a period of 1 to 12 monthswhich comprises: grinding a copolymer of lactic acid and glycolic acidhaving a ratio of glycolide to lactide units of from about 0 to 5:1 to aparticle size of between about 50 and 150 μm; wetting the ground andcopolymer with an aqueous slurry of a bioactive peptide or peptideanalog; blending the copolymer and the slurry to obtain a homogeneousmixture of the copolymer and between about 10 and 50% of the bioactivepeptide; drying the mixture at reduced pressure and at a temperature notexceeding 25° C.; extruding the dried mixture at a temperature betweenabout 70 and 110° C.; and cutting cylindrical rods of about 1 to 2 mmdiameter and between about 10 and 25 mm in length from the extrudedmixture to form the implants.

Advantageously, the ground copolymer is sterilized with a dose ofbetween about 1 and 2.5 Mrads of ionizing γ-radiation before beingcombined with the bioactive peptide, and the blending, extruding andcutting steps are conducted under aseptic conditions. Also, the implantsare generally sterilized in a conventional manner prior to beingadministered to the subject or patient.

The polymers or copolymers form a biodegradable matrix within which iscontained a uniform distribution of the peptide or peptide analog. Inthese copolymers, an advantageous ratio of glycolide to lactide unitsranges from about 0.5:1 to 3:1. One particularly preferred copolymer tobe used is soluble in benzene and has an inherent viscosity of from 0.51to 1 (1% in benzene). The amount of slurry is preferably controlled sothat the amount of water in the mixture is between about 35 and 65 ml.per 100 grams copolymer, so that the amount of bioactive peptide inthese rods is between about 10 to 50 percent by weight.

The bioactive peptide or peptide analog may be an agonist or antagonistof LHRH, GnRH, growth hormone releasing hormone, growth hormonereleasing peptide, angiotensin, bombesin, bradykin, cholecystokinin,enkephalin, neurokinin, tachykinin or substance P. The bioactive peptidemay also be an inhibitor such as a renin inhibitor, a proteaseinhibitor, a metallopeptidase inhibitor, enkephalinase and atrial orbrain natriuretic factor degrading enzyme inhibitor. The LHRH analog ispreferably a pharmaceutically acceptable salt of an LHRH agonist orantagonist, such as a pharmaceutically acceptable salt of leuprolide,goserelin, triptorelin, buserelin, avorelin, deslorelin, histrelin,cetrorelix, teverelix, ramorelix, antide, nictide, azaline B, azaline Cor ganirelix.

Another aspect of the invention relates to the pharmaceutical implantsobtained according to the process defined herein. These implants arepreferably contained in an implanter device with a retractable needle sothat they are suitable for subcutaneous injection under the skin of amammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of serum testosterone and plasma avorelin levels ofmale beagle dogs for up to 180 days after injection of the avorelinimplants of Example 1 of the invention; and

FIGS. 2 and 3 are graphs of serum LH, FSH and testosterone levels inmale patients for up to 33 to 35 weeks after injection of the avorelinimplants of Examples 2 and 3 of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Any polylactide polymer or PLGA copolymer can be used to form thebiodegradable matrix of this invention. These materials are well knownto one of ordinary skill in the art, e.g., in the U.S. patents mentionedabove, and need not be further discussed herein. The particularcopolymer is selected and then is ground to a particle size of betweenabout 50 and 150 μm. This grinding step is also conventional and needsno further explanation.

In the most preferred method, the ground copolymer is sterilized with adose of between about 1 and 2.5 Mrads of ionizing γ-radiation, again ina conventional manner that is well known to one of ordinary skill in theart.

The ground and sterilized copolymer particles are then wetted with asterile aqueous slurry of an active agent of a bioactive peptide orpeptide analog. This slurry is made by combining the peptide, analog, ora pharmaceutically acceptable salt thereof in sterile water. The amountof the active agent can vary over a wide range, from e.g. about 5 to 50and preferably about 10 to 25 grams per liter. The solution is thensterilized in a conventional manner, such as by passage through asterilizing filter. If necessary, the solution can be concentrated toincrease the amount of peptide or peptide analog therein. Theconcentration of the peptide or peptide analog in the solution can bevaried to change the resulting dosage of the implant.

Next, the copolymer and the slurry are aseptically blended to obtain ahomogeneous mixture of the copolymer and the active agent. Dependingupon the desired formulation, the active agent represents between about10 and 50% and preferably about 15 to 25% of the mixture. As notedabove, a water content of about 35 and 65 ml. and preferably about 45 to55 ml. per 100 grams copolymer in the mixture is desired. Next, themixture is dried at reduced pressure and at a temperature not exceeding25° C. to form the pharmaceutical composition. If necessary, thiscomposition can be formulated with conventional carriers as a suspensionfor injection.

Alternatively, the dried composition can be extruded with a conventionalextrusion device at a temperature between about 70 and 110° C. to form a"spaghetti" or continuous rod product. The use of heat in the extrusionstep helps further dry the product. To form the implants, thesecylindrical rods are aseptically cut into pieces of about 1 to 2 mmdiameter and between about 10 and 25 mm in length from the extrudedmixture. The length of the implant is another mechanism for varying thedosage of bioactive peptide of peptide analog therein. These productscan then be implanted subcutaneously beneath the skin of the patientusing conventional implanting devices.

The present invention provides an effective release (i.e. in terms oftherapeutic effectiveness) of a bioactive peptide, or peptide analog,such as an LHRH analog, even if such release, as measured by plasmalevel of the peptide, or peptide analog, is intermittent ordiscontinuous. This effectiveness can be achieved, for example, by theinternalization or down-regulation of pituitary receptors followingtheir exposure to LHRH agonists or to LHRH antagonists which areintrinsically long acting.

The process of this invention can be applied to a wide variety ofpeptides or peptide analogs. In addition to LHRH analogs hereinmentioned, GnRH or growth hormone releasing hormones or peptides can bementioned. Generally, any peptides or peptide analogs that arechemically stable under the process conditions and that provide asustained delivery is desirable from a therapeutic point of view.Non-limiting examples of such peptides and peptide analogs aresomatostatin and somatostatin analogs, agonist and antagonist analogs ofangiotensin II, bombesin analogs, preferably bombesin antagonists,bradykinin antagonists, preferably with minimal histamine releasingproperties, cholecystokinin analogs, preferably cholecystokininantagonists, enkephalin analogs, neurokinins, tachykinins and substanceP antagonists, renin inhibitors and other aspartyl protease inhibitors,such as HIV protease inhibitors, metallopeptidase inhibitors, such asangiotensin converting enzyme, enkephalinase and atrial or brainnatriuretic factor degrading enzyme inhibitors. The skilled artisan willfavor those peptides and peptidomimetic compounds which are not, or arepoorly, absorbed by the oral route in animals and humans, and willadjust the dose of the compound to be formulated in the implants of thepresent invention according to the biological potency of such compound,the necessary daily effective dose and the estimated duration of releasefrom the formulation.

The present invention also eliminates contamination of such formulationswith organic solvents, particularly chlorinated ones, such as chloroformor methylene chloride, which are typically utilized in the manufactureof microspheres or microcapsules by the coacervation-solvent evaporationmethods (see, e.g., U.S. Pat. No. 3,773,919) or which are used tosterilize PLGA copolymers by filtration.

The present invention does not make use of any organic solvent, buttakes advantage of the unorthodox use of water, a solvent hithertoconsidered unsuitable for such formulations because of its deleteriouseffect on the polyester (copolymer) of the PLGA matrix, where it canaccelerate chemical hydrolysis and also damage structural integrity uponexposure to ionizing radiation (formation of free radicals) during thesterilization step necessary for safety considerations.

Another advantage of this unorthodox use of water is to achieve auniform coating of the active principle on the granulated polymerpowder, resulting in a much needed and highly desired uniformity of themixture, an essential condition of the manufacturing process. A furtherunexpected advantage of this unconventional solvent is the "wettability"of the powdery mixture which would otherwise create serious problems dueto formation of static electrical charges which can cause unacceptablemechanical losses and loss of uniformity.

The instant process further provides with a simple method ofsterilization of the composition by subjecting the polymer to ionizingradiation prior to blending the polymer with the bioactive peptides orpeptide analogs which are invariably damaged by such radiation,resulting in unwanted byproducts. A further advantage of the instantprocess is to provide a variable sterilizing dose of radiation (from 1to 2.5 Mrad) predetermined by the actual biomass present in theco-polymer, with a resulting safety without undue creation of radiolysisartifacts.

EXAMPLES

The following examples are submitted to illustrate the effectiveness ofthe most preferred formulations of the invention.

Example 1

The manufacturing process is conducted in a commercially availableisolator (ARFL, Neuilly-sur-Marne, France) equipped with air-locks forthe introduction of pre-sterilized components and itself sterilized byprevious peracetic acid treatment. The extrusion machine is acommercially available single screw extruder (Brabender, 47055 Duisburg,Germany) equipped with pressure and temperature probes. The cuttingmachine is commercially available (Davis-Standard Corp. Cedar Grove,N.J., USA). Blenders/mixers and weighing instruments are conventionalequipment.

A quantity of 80 g of racemic lactic acid and glycolic acid copolymer(75:25) soluble in benzene and of inherent viscosity of 0.60 (1% inbenzene) (PuracBiochem B. V., Gorinchem, Netherlands) is ground andsieved to collect the fraction of particles between 50 to 150 μm andsterilized with an ionizing γ-radiation of 1.5 Mrads by a commerciallaboratory (Caric-Mediris, Fleurus, Belgium) and introduced through theair-lock into the sterile isolator.

Separately, 23 grams of the LHRH analog avorelin acetate (INN), or(2Methyl-D-Trp)⁶ (des-Gly)¹⁰ (ProEthylamide)⁹ LHRH acetate, dissolved in500 ml of sterile water and filtered through a Millipore 0.2 μmsterilizing filter. The sterile solution is reduced by evaporation to avolume of 50 ml and the resulting mixture is dispersed through theground co-polymer. The wet mixture is blended to obtain a granulatecontaining 20% of avorelin. Such mixture is dried at 25° C. underreduced pressure and then extruded at a temperature gradient from 70 to110° C. at pressures of 3500 p.s.i. The resulting extrudate isaseptically cut to give rods of 1.5 mm diameter and 15 mm long,containing 10 mg avorelin, which are inserted into a pre-sterilizedimplanter with a retractable needle (SFM GmbH, D-6480 Wachtersbach,Germany) sealed and used as such, or optionally further sterilized witha dose of 1.5 Mrad of γ-radiation before clinical use.

When implanted s.c. into male beagle dogs, after the initial stimulationof LH and testosterone, castration levels of testosterone weremaintained for 6 months. The plasma levels of avorelin, after ashort-lived burst, fell to a nadir at 40 days and rose again at 120 daysbefore becoming undetectable at day 160. These results are shown in FIG.1.

Example 2

Following essentially the procedure of Example 1, 10 mg avorelinimplants were prepared, further sterilized, and implanted into healthymale patients. After initial stimulation of LH, FSH and testosterone,these levers were significantly reduced, with the testosterone levelbeing maintained below a castration level for 33 weeks. These resultsare shown in FIG. 2.

Example 3

Following essentially the procedure of Example 2, except that the lengthof the implant was increased to provide a dose of avorelin of 15 mg wasprepared. These implants were sterilized and implanted into healthy malepatients. After initial stimulation of LH, FSH and testosterone, theselevels were significantly reduced, with the testosterone level beingmaintained below a castration level for 33 weeks. These results areshown in FIG. 3.

Example 4

Following essentially the procedure of Example 1 with appropriatemodifications required by the individual LHRH analog, rods containing 22mg of leuprolide, 10 mg of goserelin and 30 mg of teverelix weresimilarly obtained.

What is claimed is:
 1. A process for manufacturing pharmaceutical implants for the delivery of an effective amount of a bioactive peptide or peptide analog over a period of 1 to 12 months which comprises:grinding a copolymer of lactic acid and glycolic acid having a ratio of glycolide to lactide units of from about 0 to 5:1 to a particle size of between about 50 and 150 μm; wetting said ground and sterilized copolymer with a sterile aqueous slurry of a bioactive peptide or peptide analog; blending the copolymer and the slurry to obtain a homogeneous mixture of said copolymer and between about 10 and 50% of the bioactive peptide or peptide analog; drying said mixture at reduced pressure and at temperature not exceeding 25° C.; extruding said dried mixture at a temperature between about 70 and 110° C.; and cutting cylindrical rods of about 1 to 2 mm diameter and between about 10 and 25 mm in length from the extruded mixture to form the pharmaceutical implants.
 2. The process of claim 1 which further comprises sterilizing said ground copolymer with a dose of between about 1 and 2.5 Mrads of ionizing γ-radiation before adding the aqueous slurry thereto.
 3. The process of claim 1 which further comprises conducting the blending, extruding and cutting steps are conducted aseptically.
 4. The process of claim 1 which further comprises selecting the copolymer to be used to be one which is soluble in benzene and has an inherent viscosity of from 0.51 to 1 (1% in benzene).
 5. The process of claim 1 wherein the amount of slurry is controlled so that the amount of water in the mixture is between about 35 and 65 ml. per 100 grams copolymer.
 6. The process of claim 1 wherein the amount of slurry is controlled so that the amount of bioactive peptide or peptide analog in the rods is between about 10 to 50 percent by weight.
 7. The process of claim 1 wherein the ratio of glycolide to lactide units in the copolymer ranges from about 0.5:1 to 3:1.
 8. The process of claim 1 wherein the bioactive peptide or peptide analog is an agonist or antagonist of LHRH, GnRH, growth hormone releasing hormone, growth hormone releasing peptide, angiotensin, bombesin, bradykin, cholecystokinin, enkephalin, neurokinin, tachykinin or substance P.
 9. The process of claim 1 wherein the bioactive peptide or peptide analog is a renin inhibitor, a protease inhibitor, a metallopeptidase inhibitor, enkephalinase and atrial or brain natriuretic factor degrading enzyme inhibitor.
 10. The process of claim 8 wherein the bioactive peptide or peptide analog is a pharmaceutically acceptable salt of leuprolide, goserelin, triptorelin, buserelin, avorelin, deslorelin, histrelin, cetrorelix, teverelix, ramorelix, antide, nictide, azaline B, azaline C or ganirelix. 